Dioxygenases catalyze the incorporation of both atoms of molecular oxygen into a substrate. In bacteria, this ring-opening reaction is a key step in the degradation pathway for many aromatic compounds found in the environment. In plants and animals, dioxygenases are involved in the metabolism of indoles, phenylalanine, arachidonic acids and prostaglandins. Dioxygenases are typically metalloproteins many of which require non-heme mononuclear iron center as a cofactor. This project has been undertaken to discover the structural foundation for catalysis in these enzymes. The first structure of a dioxygenase was determined by the P.I. This metalloenzyme, protocatechuate 3,4-dioxygenase (3,4-PCD) from Pseudomonas aeruginosa (Pa-PCD), has been refined to an R-value of 0.172 to 2.15 Angstroms resolution. Pa-PCD has been cloned and expressed; a number of mutants have been made. Diffraction data has been collected on five Pa- PCD:inhibitor complexes, three of which are under refinement (current R- values <0.20 to at least 2.5 Angstroms resolution). The structure of 3,4- PCD from Acinetobacter calcoaceticus (Ac-PCD) has been solved and is under refinement (current R-value of 0.219 to 2.5 Angstroms resolution). Several mutants of Ac-PCD are known. Crystals have been grown and diffraction data collected from catechol 1,2-dioxygenase (1,2-CTD) from P. arvilla and from 3,4-PCD from the gram positive organism Brevibacterium fuscum. In addition, crystals of catechol 2,3-dioxygenase and benzoate 1,2- dioxygenase from P. arvilla have been grown. The initial target of the proposal is 3,4-PCD which has been studied extensively using a number of spectroscopic techniques over the past three decades. The structural data will be combined not only with activity assays but also with a wide variety of spectroscopic, kinetic and genetic techniques available in the laboratories of our collaborators. This combined approach will allow a number of questions to be addressed including: How does iron ligation change during catalysis? What is the role of the ligands in binding and preparing the iron for catalysis? What is the basis of substrate specificity? What are the functional consequence of varying protomer stoichiometry? Why were active sites discarded by evolution? And, how can activity be restored to these vestigial active sites?